The class of compounds known as polyketides is a large family of diverse compounds synthesized primarily from 2-carbon unit building block compounds through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria such as the actinomycetes. There are a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. Epothilone, erythromycin, FK-506, FK-520, megalomicin, narbomycin, oleandomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of such compounds.
Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds. See PCT Publication Nos. WO 95/08548; WO 96/40968; WO 97/02358; and 98/27203; U.S. Pat. Nos. 5,962,290; 5,672,491; and 5,712,146; Fu et al., Biochemistry 33: 9321-9326 (1994); McDaniel et al, Science 262:1546-1555 (1993); and Rohr, Angew. Chem. Int. Ed. Engl. 34(8): 881-888 (1995), each of which is incorporated herein by reference.
Polyketides are synthesized in nature by polyketide synthase (PKS) enzymes. These enzymes, which are complexes of multiple large proteins, are similar to the synthases that catalyze condensation of 2-carbon unit building block compounds in the biosynthesis of fatty acids. The genes that encode PKS enzymes usually consist of three or more open reading frames (ORFs). Two major types of PKS enzymes are known that differ in their composition and mode of synthesis. These two major types of PKS enzymes are commonly referred to as Type I or “modular” and Type II or “iterative” PKS enzymes.
Modular PKSs produce many different polyketides, including a large number of 12-, 14-, and 16-membered macrolide antibiotics including erythromycin, megalomicin, methymycin, narbomycin, oleandomycin, picromycin, and tylosin. Each ORF of a modular PKS can comprise one, two, or more “modules” of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three (for the simplest extender module) or more enzymatic activities or “domains.” These large multifunctional enzymes (>300,000 kDa) catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying β-carbon processing activities (see O'Hagan, D., The polyketide metabolites, E. Horwood, New York, 1991, which is incorporated herein by reference).
During the past half decade, the study of modular PKS function and specificity has been greatly facilitated by the plasmid-based Streptomyces coelicolor expression system developed with the 6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al. Science, 265: 509-512 (1994), McDaniel et al., Science 262: 1546-1557 (1993), and U.S. Pat. Nos. 5,672,491 and 5,712,146, each of which is incorporated herein by reference). The advantages to this plasmid-based genetic system for DEBS are that it overcomes the tedious and limited techniques for manipulating the natural DEBS host organism, Saccharopolyspora erythraea, allows more facile construction of recombinant PKSs, and reduces the complexity of PKS analysis by providing a “clean” host background. This system also expedited construction of a combinatorial modular polyketide library in Streptomyces (see PCT publication No. WO 98/49315, incorporated herein by reference).
The ability to control aspects of polyketide biosynthesis, such as monomer selection and degree of β-carbon processing, by genetic manipulation of PKSs has stimulated great interest in the combinatorial engineering of novel antibiotics (see Hutchinson, Curr. Opin. Microbiol. 1: 319-329 (1998); Carreras and Santi, Curr. Opin. Biotech. 9: 403-411 (1998); and U.S. Pat. Nos. 5,962,290; 5,712,146; and 5,672,491, each of which is incorporated herein by reference). This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes. The resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters.
Polyketides are assembled by polyketide synthases through successive condensations of activated coenzyme-A thioester monomers derived from small organic acids such as acetate, propionate, and butyrate. Active sites required for condensation include an acyltransferase (AT), acyl carrier protein (ACP), and beta-ketoacylsynthase (KS). Each condensation cycle results in a β-keto group that undergoes all, some, or none of a series of processing activities. Active sites that perform these reactions include a ketoreductase (KR), dehydratase (DH), and enoylreductase (ER). Thus, the absence of any beta-keto processing domain results in the presence of a ketone, a KR alone gives rise to a hydroxyl, a KR and DH result in an alkene, while a KR, DH, and ER combination leads to complete reduction to an alkane. After assembly of the polyketide chain, the molecule typically undergoes cyclization(s) and post-PKS modification (e.g. glycosylation, oxidation, acylation) to achieve the final active compound.
To illustrate the synthesis of a macrolide by a modular PKS (see Cane et al., Science 282: 63 (1998), incorporated herein by reference), one can refer to the PKS that produces the erythromycin polyketide (6-deoxyerythronolide B synthase or DEBS; see U.S. Pat. No. 5,824,513, incorporated herein by reference). In the modular DEBS PKS enzyme, the enzymatic steps for each round of condensation and reduction are encoded within a single “module” of the polypeptide (i.e., one distinct module for every condensation cycle). As shown in FIG. 1, DEBS consists of a loading module and 6 extender modules and a chain terminating thioesterase (TE) domain within three extremely large polypeptides encoded by three open reading frames (ORFs, designated eryAI, eryAII, and eryAIII).
Each of the three polypeptide subunits of DEBS (DEB1, DEBS2, and DEBS3 in FIG. 1) contains 2 extender modules. DEBS1 additionally contains the loading module, and DEBS3 contains the TE domain. Collectively, these proteins catalyze the condensation and appropriate reduction of one propionyl CoA starter unit and six methylmalonyl CoA extender units. Modules 1, 2, 5, and 6 contain KR domains; module 4 contains a complete set, KR/DH/ER, of reductive and dehydratase domains; and module 3 contains no functional reductive domain. Following the condensation and appropriate dehydration and reduction reactions, the enzyme bound intermediate is lactonized by the TE at the end of extender module 6 to form 6-dEB (compound 1 in FIG. 1).
More particularly, the loading module of DEBS consists of two domains, an acyl-transferase (AT) domain and an acyl carrier protein (ACP) domain. In other PKS enzymes, the loading module is not composed of an AT and an ACP but instead utilizes a partially inactivated KS, an AT, and an ACP. This partially inactivated KS is in most instances called KSQ, where the superscript letter is the abbreviation for the amino acid, glutamine, that is present instead of a cysteine in the active site that is believed to be required for condensation activity. Although the KSQ domain lacks condensation activity, it retains decarboxylase activity. The AT domain of the loading module recognizes a particular acyl-CoA (propionyl for DEBS, which can also accept acetyl) and transfers it as a thiol ester to the ACP of the loading module. Concurrently, the AT on each of the extender modules recognizes a particular extender-CoA (methylmalonyl for DEBS) and transfers it to the ACP of that module to form a thioester. Once the PKS is primed with acyl- and malonyl-ACPs, the acyl group of the loading module migrates to form a thiol ester (trans-esterification) at the KS of the first extender module; at this stage, extender module 1 possesses an acyl-KS and a methylmalonyl ACP. The acyl group derived from the loading module is then covalently attached to the alpha-carbon of the malonyl group to form a carbon-carbon bond, driven by concomitant decarboxylation, and generating a new acyl-ACP that has a backbone two carbons longer than the loading unit (elongation or extension). The growing polyketide chain (various intermediates are shown in FIG. 1) is transferred from the ACP to the KS of the next module, and the process continues.
The polyketide chain, growing by two carbons each module, is sequentially passed as a covalently bound thiol ester from module to module, in an assembly line-like process. The carbon chain produced by this process alone would possess a ketone at every other carbon atom, producing a polyketone, from which the name polyketide arises. Commonly, however, additional enzymatic activities modify the beta keto group of the polyketide chain to which the two carbon unit has been added before the chain is transferred to the next module. Modules may contain additional enzymatic activities as well, such as methyl transferase domains, but there are no such additional activities in DEBS.
Once a polyketide chain traverses the final extender module of a modular PKS, it encounters the releasing domain or thioesterase found at the carboxyl end of most PKSs. Here, the polyketide is cleaved from the enzyme and cyclyzed. The resulting polyketide can be modified further by tailoring or modification enzymes; these enzymes add carbohydrate groups or methyl groups, or make other modifications, i.e., oxidation or reduction, on the polyketide core molecule. For example, the final steps in conversion of 6-dEB to erythromycin A include the actions of a number of modification enzymes, such as: C-6 hydroxylation, attachment of mycarose and desosamine sugars, C-12 hydroxylation (which produces erythromycin C), and conversion of mycarose to cladinose via O-methylation. These modifications in various combinations result in erythromycins A (compound 2 in FIG. 1), B, C, and D.
While the detailed understanding of the mechanisms by which PKS enzymes function and the development of methods for manipulating PKS genes have facilitated the creation of novel polyketides, there remain substantial impediments to the creation of novel polyketides by genetic engineering. One such impediment is the availability of PKS genes. Many polyketides are known but only a relatively small portion of the corresponding PKS genes have been cloned and are available for manipulation. Moreover, in many instances the producing organism for an interesting polyketide is obtainable only with great difficulty and expense, and techniques for its growth in the laboratory and production of the polyketide it produces are unknown or difficult or time-consuming to practice. Also, even if the PKS genes for a desired polyketide have been cloned, those genes may not serve to drive the level of production desired in a particular host cell.
If there were a method to produce a desired polyketide without having to access the genes that encode the PKS that produces the polyketide, then many of these difficulties could be ameliorated or avoided altogether. The present invention meets this need.